Cuticle protein genes are comprised of 2 or 3 multigene families that are differentially expressed in response to the steroid hormone, 20-hydroxyecdysone (20HOE) during Drosophila development. Genes for larval cuticle proteins are expressed in the larval epidermis. Genes for pupal cuticle proteins, related to but distinct from the larval ones, are expressed in imaginal discs in prepupae. Expression of the pupas genes is induced in discs by 20HOE in vitro. Genomic clones containing a cluster of four 3rd instar cuticle protein genes have been recovered and characterized (Snyder et al., 1981) (35). Variants affecting the expression of these genes have been recovered. We propose to clone the genes for the pupal cuticle proteins and produce polyclonal and monoclonal antibody against these proteins. These reagents will be used to describe the molecular details of the onset of synthesis, in response to 20HOE, of cuticle proteins and their mRNAs in imaginal discs in vitro. We will systematically recover variants affecting the expression of larval and pupal cuticle genes. Both "cis"-acting and "distant regulators" of gene expression will be recovered and characterized genetically and at the DNA level. Pupal cuticle gene variants will be analyzed in discs cultured in vitro to determine the molecular nature of the alteration in expression using the cloned genes and the antibody to monitor synthesis. New information about the regulation of gene expression during development should result from these studies.